Facioscapulohumeral dystrophy (FSHD) is one of the most commonly inherited muscular dystrophies in the United States. It is characterized by progressive weakness and atrophy of facial, shoulder, and upper arm musculature, which can spread to the abdominal and foot-extensor muscles. The genetics underlying FSHD are highly unusual; most cases (>95%) of FSHD involve mono-allelic deletion of macrosatellite D4Z4 repeat sequences at the subtelomeric region of chromosome 4q (FSHD1), while the remaining ~5% of cases demonstrate no D4Z4 repeat contraction (FSHD2). The majority of FSHD2 is linked to mutations of SMCHD1, which is known to be involved in a certain aspect of the maintenance of DNA methylation though its detailed function remains unclear. Recent studies argue that the derepression of the DUX4 gene encoded within the D4Z4 repeat is critically linked to the development of FSHD. Exactly how the DUX4 gene is upregulated in patient muscle cells, however, is not well understood. We previously found that the D4Z4 repeat cluster normally contains a transcriptionally repressive heterochromatin structure containing histone H3 lysine 9 trimethylation (H3K9me3), H3K27me3, heterochromatin protein 1 gamma (HP1?), and the higher-order chromatin organizer cohesin. Interestingly, H3K9me3 is specifically diminished at D4Z4 in both FSHD1 and FSHD2 patient cells, which also results in the reduction of HP1? and cohesin binding in this region. This change appears to be unique to FSHD and not to other muscular dystrophies, suggesting that FSHD is a heterochromatin abnormality disease. We recently obtained data showing that inhibition of H3K9me3 at D4Z4 increases DUX4 expression, indicating that the loss of D4Z4 heterochromatin indeed affects gene expression and contributes to FSHD pathogenesis. However, how H3K9me3 at D4Z4 is regulated and contributes to gene regulation and how it is diminished in both FSHD1 and FSHD2 are currently unclear. Thus, the goal of this project is to characterize D4Z4 chromatin regulation and generate FSHD-modeling human myoblast lines to delineate the disease mechanism. Primary control and patient myoblast samples are important resources to study FSHD since there is no clearly defined mouse model available (there are no D4Z4 repeats in mice). However, the number of cells that can be obtained from painful muscle biopsies is limited, and thus it is pertinent to establish models that recapitulate FSHD conditions. An immortalized human myoblast line that maintains high proliferation and differentiation capabilities is an ideal system to study human myogenesis. In this project, we plan to use this cell line to (1) systematically identify the components of D4Z4 heterochromatin, and (2) create FSHD-modeling lines by modulating the D4Z4 copy number, SMCHD1 expression and H3K9me3 level at D4Z4. The successful outcome of this project may lead to further understanding of the disease mechanism(s) and identification of potential new therapeutic targets and approaches for treatment.